Theoretical studies of cyclic adenosine monophosphate dependent protein kinase: native enzyme and ground-state and transition-state analogues.

The mechanisms of phosphoryl transfer enzymes have garnered considerable attention. Cyclic AMP-dependent protein kinase (cAPK) catalyzes the transfer of the γ phosphoryl group of ATP to the serine hydroxyl group of a peptide chain. Metal-containing fluoro species have been used as transition-state and ground-state analogues in a variety of phosphoryl transfer enzymes and have shed light on the nature of the requirements in the active site to catalyze phosphoryl transfer. For cAPK, we present computational studies of the mechanism of phosphoryl transfer and the structure and (19)F NMR spectra of various ground- (BeF3(-)) and transition-state (MgF3(-), AlF4(-), and AlF3(0)) analogues. With native substrate, the phosphoryl transfer proceeds through a five-coordinate phosphorane transition state, i.e., there is not a five-coordinate phosphorane intermediate. Comparisons of simulated and experimental (19)F NMR spectra show cAPK prefers a monoanionic analogue (MgF3(-) or AlF4(-)) over a neutral analogue (AlF3), supporting the charge balance hypothesis.

Near attack conformers dominate ß-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate.

Experimental observations of fluoromagnesate and fluoroaluminate complexes of ß-phosphoglucomutase (ß-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of ß-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of ß-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.